Methods and Materials for the Detection of Leishmania Infection

ABSTRACT

The present invention provides rapid diagnostic assays for the detection of  Leishmania  which can readily be used in the field, leading to more rapid treatment. In certain embodiments, the inventive assays, including ELISA and lateral flow assays, employ antibodies that may be effectively employed to detect the  Leishmania major  antigen TSA, which is present in both promastigotes grown in culture and in amastigotes. Such assays may be employed to detect the presence of cutaneous leishmaniasis in a subject using scrapings, biopsies, and/or aspirates taken from cutaneous lesions.

REFERENCE TO PRIORITY APPLICATION

This application claims priority to U.S. provisional patent applicationNo. 60/742,761, filed Dec. 6, 2005.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant No.W81XWH-05-C-0018 awarded by the Department of Defense.

FIELD OF THE INVENTION

The present invention relates generally to the diagnosis of Leishmaniainfection. More specifically, the invention relates to the immunologicaldetection of cutaneous leishmaniasis.

BACKGROUND OF THE INVENTION

Leishmania organisms are intracellular protozoan parasites ofmacrophages that cause a wide range of clinical diseases in humans anddomestic animals, primarily dogs. The life cycles of Leishmania involvesa vertebrate host (e.g., a human) and a vector (a sand fly) thattransmits the parasite between vertebrate hosts. In the vector theparasite takes on a characteristic morphological form known as thepromastigote, and reproduces asexually in the vector's gut. When thevector bites a vertebrate host, promastigotes are injected into thehost. The promastigotes then enter cells of the vertebrate host andchange into a form known as the amastigote. The amastigote reproduces inthe host's cells and, when the cells eventually die, the amastigotes arereleased and infect other cells. The symptoms and pathology associatedwith leishmaniasis result from the amastigotes killing the host's cells.

In some infections, the parasite may lie dormant for many years. Inother cases, the host may develop one of a variety of forms ofleishmaniasis. For example, leishmaniasis may be manifested as acutaneous disease, which is a severe medical problem. Several Leishmaniaspecies are responsible for cutaneous leishmaniasis. In the Middle Eastand Central Asia the predominant species responsible for cutaneous formsof leishmaniasis are L. major and L. tropica, with L. donovani and L.infantu predominantly leading to visceral forms of leishmaniasis. InIraq L. major is the major cause of cutaneous leishmaniasis with most ofthe reported cases in the army being due to this agent. L. major is theprimary agent for zoonotic cutaneous leishmaniasis (ZCL). In Afghanistanthe primary agent for cutaneous leishmaniasis is L. tropica with anactive infection rate in Kabul of 2.7%. However areas of NorthernAfghanistan are also endemic for L. major. L. tropica is more frequentlyassociated with anthroponotic cutaneous leishmaniasis (AZL). With thedeployment of U.S. troops to the Middle East and Central Asia there hasbeen a significant increase in the number of personnel developingcutaneous leishmaniasis, particularly in Iraq. This has raised the needfor a field test to identify the presence of Leishmania parasitesdirectly in skin lesions.

Current diagnostic procedures are not readily applicable to fieldsituations and typically require centralized laboratory testing. Inparticular PCR has gained a forefront in testing for Leishmania speciesinvolved in cutaneous leishmaniasis. Material from skin scrapings orbiopsy is obtained, and DNA extracted and subjected to PCR analysis. NewPCR methods are being developed to enable multiple Leishmania species tobe detected and differentiated in a single assay. Other methods used foridentifying parasites include culture from skin biopsy samples, which istime consuming, or light microscopic analysis or histology of thintissue sections from lesions or biopsies to analyze for the presence ofparasites. In the case of visceral leishmaniasis, a rapid diagnostictest is available based on use of the repeat antigen K39 to detect thepresence of antibodies to L. donovani, L. chagasi and L. infantu inserum (Scott et al., Am. J. Trop. Med. Hyg., 44:272-277, 1991). Asimilar assay does not currently exist for Leishmania species involvedin cutaneous leishmaniasis.

Serodiagnosis looking for specific antibodies in cutaneous leishmaniasishas been attempted with some success using secreted antigens but thistest lacks sufficient sensitivity and specificity. More recently, lessinvasive procedures have been used involving aspirates or scrapings fromskin lesions or by using swabs (see, for example, Matsumoto et al.,Trans. R. Soc. Trop. Med. Hyg. 93:606-7, 1999). These procedures lendthemselves for use as collection devices for rapid field tests. Rabbitanti gp63 has been used in an indirect immunofluorescence assay fordetection of Leishmania promastigotes and amastigotes in lesionaspirates (Mohareb et al., J. Egypt Soc. Parasitol. 28:313-321, 1998).Analysis of Western blot banding patterns has been used in thedifferential diagnosis of American cutaneous leishmaniasis (Goncalves etal., Am. J. Trop. Med. Hyg. 66:91-102, 2002). Skin tests usingLeishmanin have also been used to evaluate latent infection but havegenerally been based on crude lysate antigen preparations and lackspecificity (Arana et al., Trans. R. Soc. Trop. Med. Hyg. 93:394-6,1999).

There thus remains a need in the art for a rapid and effectivediagnostic test for cutaneous leishmaniasis that may be readily employedin a field situation.

SUMMARY OF THE INVENTION

The present invention provides rapid diagnostic assays for the detectionof Leishmania amastigotes, promastigotes and/or secreted proteins whichcan readily be used in the field, leading to more rapid treatment. Inone embodiment, the present invention provides diagnostic tests,including ELISA and lateral flow assays, that may be effectivelyemployed to detect a Leishmania major antigen that is present in bothpromastigotes grown in culture and, more importantly, in amastigotes—theform present in cutaneous lesions. Such assays may be employed to detectthe presence of cutaneous leishmaniasis in a subject using scrapings,biopsies, and/or aspirates taken from cutaneous lesions. In anotherembodiment, diagnostic tests, including ELISA and lateral flow assays,are provided that may be effectively employed to detect a Leishmaniamajor antigen that is present in secreted proteins and promastigotes butnot in amastigotes. Such assays may be employed to detect the presenceof cutaneous leishmaniasis in a subject using biological samples such asserum, and scrapings, biopsies, and/or aspirates taken from cutaneouslesions

In certain embodiments, the assays disclosed herein employ antibodiesspecific for the Thiol Specific Antigen (TSA also known as peroxidoxin;SEQ ID NO: 1 and 2). Preferably the antibodies employed in such assaysbind to a conformational epitope of TSA that is present in amastigotes.In one embodiment, the assays employ a first TSA-specific antibody as acapture antibody, with a second TSA-specific antibody being employed asa detection antibody. Preferably, the detection antibody is labeled witha reporter group or agent. For example, a monoclonal antibody againstTSA, such as the antibody C11C (which was initially raised againstamastigotes), may be employed as the capture antibody, with a rabbitpolyclonal antibody to recombinant TSA being employed as the detectionantibody. In the case of an ELISA, further development of the assay maythen be performed using a goat anti-rabbit IgG horseradish peroxidaselabel. For a lateral flow assay, the anti-TSA antibody is preferablylabeled with a calorimetric or fluorescent indicator, such as colloidalgold or a fluorescent dye, thereby allowing a user to determine visuallywhether a test is positive or negative for cutaneous leishmaniasis.

Dipsticks for use in such lateral flow assays are also provided. Incertain embodiments, such dipsticks comprise: (a) a lateral flowmembrane; (b) a first area positioned at a first, lower, end of thelateral flow membrane for receiving a test sample, wherein the firstarea comprises a first antibody specific for a polypeptide of SEQ ID NO:1, the antibody being labeled with a reporter agent; (c) a second areapositioned at a second, upper, end of the lateral flow membranecomprising an immobilized control polypeptide; and (d) a third areapositioned between the first and second areas, wherein the third areacomprises an immobilized second antibody specific for a polypeptide ofSEQ ID NO: 1.

Diagnostic kits comprising such dipsticks are also provided. In certainembodiments, such diagnostic kits comprise a dipstick, a vesselcontaining lysing buffer, a rod for mixing a biological sample with thebuffer to provide a test solution, and a pipette for applying the textsolution to the dipstick. Preferably such kits are sealably containedwithin a container, such as an aluminum pouch, that is generallyimpermeable to gases and fluids.

In alternative embodiments, the assays for the detection of L. majorinfection disclosed herein employ an antibody specific for L. majorlipophosphoglycan (LPG), such as the monoclonal antibody WIC79.3. Suchassays may be in the form of an ELISA or a lateral flow assay asdetailed below. Dipsticks for use in such lateral flow assays, anddiagnostic kits comprising such dipsticks are also provided.

The above-mentioned and additional features of the present invention andthe manner of obtaining them will become apparent, and the inventionwill be best understood by reference to the following more detaileddescription. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a dipstick for use in a lateral flow assay of the presentinvention.

FIGS. 2A and B show the reactivity of the antibody C11C againstdifferent concentrations of both promastigotes and amastigotes asdetermined by ELISA.

FIGS. 3-5 show the reactivity of various antibodies againstpromastigotes and amastigotes obtained from mouse skin lesions asdetermined by ELISA.

FIG. 6 shows a standard curve for detection of TSA using a sandwichELISA employing the antibody C11C as the capture antibody and anaffinity purified rabbit anti-TSA antibody as the detection antibody.LACK antigen was used as a negative control.

FIG. 7 shows the reactivity of additional monoclonal antibodies torecombinant TSA.

FIGS. 8A and 8B show the ability of the assay of FIG. 6 to detect TSA incultured L. major promastigotes as well as L. major and L. mexicanaamastigotes from infected mice but not in E. coli lysate and exoantigensfrom various Leishmania spp.

FIG. 9 shows the reactivity of a lateral flow assay for TSA and itsability to detect low cell numbers of protein emanating from amastigotesof L. major as well as recombinant TSA but not with exoantigens ofvarious Leishmania spp.

FIG. 10 shows the ability of the monoclonal antibody WIC79.3 to detectL. major secreted proteins as well as whole L. major promastigotes.

FIG. 11 shows the ability of a lateral flow assay employing the antibodyWIC79.3 to detect L. major secreted proteins, or exoantigens, but not L.tropica or L. mexicana in the same concentration range.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides methods and materialsfor detecting cutaneous leishmaniasis (CL) infection in a biologicalsample, such as scrapings, biopsies, and/or aspirates, taken fromcutaneous lesions of individuals, such as humans and/or other mammals,suspected of being infected with Leishmania. The presence of CLinfection may be detected using one or more of the assays describedherein to determine the presence or absence of a Leishmania amastigoteantigen in a sample. Preferably, the antigen detected using theinventive methods and materials is Thiol Specific Anti-oxidant proteinfrom L. major (TSA; also known as peroxidoxin; Webb et al., Infect.Immun. 66:3279-89, 1998). TSA has previously been shown to induceprotection against CL infection in a primate model (Campos-Neto et al.,Infect. Immun. 69:4103-4108, 2001). The amino acid sequence for TSA isprovided in SEQ ID NO: 1, with the cDNA sequence being provided in SEQID NO: 2. In certain embodiments, the inventive assays employantibodies, either monoclonal or polyclonal, specific for TSA to detectthe presence of Leishmania amastigotes in a biological sample.Preferably the antibodies employed in the assays are specific for aconformational epitope of TSA present in amastigotes.

In an alternative embodiment, the present invention provides methods andmaterials for detecting cutaneous leishmaniasis in a biological sample,such as serum and/or scrapings, biopsies, and/or aspirates taken fromcutaneous lesions of individuals, such as humans and/or other mammals,suspected of being infected with Leishmania. In this embodiment, anantibody, such as the monoclonal antibody WIC79.3, is used to detect thepresence of an antigen present in L. major secreted proteins and/orpromastigotes, but not in L. major amastigotes.

There are a variety of assay formats known to those of ordinary skill inthe art for using antibodies to detect an antigen in a sample which maybe effectively employed in the inventive methods. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. In one such assay format, an antibody, such as a TSA-specificantibody (referred to as the capture antibody) is immobilized on a solidsupport (as described below) and contacted with the sample to be tested.After removal of the unbound sample, a second TSA-specific antibody(referred to as the detection antibody), which has been labeled with areporter group, may be added and used to detect bound antigen.Alternatively, such assays may employ the monoclonal antibody WIC79.3.

In an exemplary competitive assay, the sample may be combined witheither a monoclonal or polyclonal antibody, which has been labeled witha suitable reporter group. The mixture of sample and antibody may thenbe combined with polypeptide antigen immobilized on a suitable solidsupport. Antibody that has not bound to an antigen in the sample isallowed to bind to the immobilized antigen, and the remainder of thesample and antibody is removed. The level of antibody bound to the solidsupport is inversely related to the level of antigen in the sample.Thus, a lower level of antibody bound to the solid support indicates thepresence of Leishmania infection in the sample. To determine thepresence or absence of Leishmania infection, the signal detected fromthe reporter group that remains bound to the solid support is generallycompared to a signal that corresponds to a predetermined cut-off value.Such cut-off values may generally be determined as described below. Anyof the reporter groups discussed below may be used to label theantibodies, and binding may be detected by any of a variety oftechniques appropriate for the reporter group employed.

In one embodiment, the inventive assay involves the use of antibodyimmobilized on a solid support to bind to and remove the antigen fromthe sample. The bound antigen may then be detected using a detectionreagent that binds to the antigen/antibody complex and contains adetectable reporter group. Suitable detection reagents includeantibodies that bind to the antigen/antibody complex labeled with areporter group. Alternatively, a competitive assay may be utilized, inwhich an antibody that binds to the antigen is labeled with a reportergroup and allowed to bind to the immobilized antigen after incubation ofthe antibody with the sample. The extent to which components of thesample inhibit the binding of the labeled antibody to the antigen isindicative of the reactivity of the sample with the immobilizedantibody.

The solid support may be any solid material known to those of ordinaryskill in the art to which the fusion polypeptide may be attached. Forexample, the solid support may be a test well in a microtiter plate, ora nitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, formed of glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681.

The antibody may be bound to the solid support using a variety oftechniques known to those in the art, which are amply described in thepatent and scientific literature. In the context of the presentinvention, the term “bound” refers to both noncovalent association, suchas adsorption, and covalent attachment (which may be a direct linkagebetween the antigen and functional groups on the support or may be alinkage by way of a cross-linking agent). Binding by adsorption to awell in a microtiter plate or to a membrane is preferred. In such cases,adsorption may be achieved by contacting the polypeptide, in a suitablebuffer, with the solid support for a suitable amount of time. Thecontact time varies with temperature, but is typically between about 1hour and 1 day. In general, contacting a well of a plastic microtiterplate (such as polystyrene or polyvinylchloride) with an amount offusion polypeptide ranging from about 10 ng to about 1 μg, andpreferably about 100 ng, is sufficient to bind an adequate amount ofantigen. Nitrocellulose will bind approximately 100 μg of protein percm³.

Covalent attachment of the antibody to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the antibody. For example, the antibody maybe bound to supports having an appropriate polymer coating usingbenzoquinone or by condensation of an aldehyde group on the support withan amine and an active hydrogen on the antibody (see, e.g., PierceImmunotechnology Catalog and Handbook (1991) at A12-A13).

In certain embodiments, the assay is an enzyme linked immunosorbentassay (ELISA). This assay may be performed by first immobilizing anantibody (referred to as the capture antibody) on a solid support,commonly the well of a microtiter plate. The immobilized antibody isthen incubated with the biological sample, and antigen (if present inthe sample) is allowed to bind to the antibody. The sample may bediluted with a suitable diluent, such as phosphate-buffered saline (PBS)prior to incubation. In general, an appropriate contact time (i.e.,incubation time) is that period of time that is sufficient to detect thepresence of Leishmania amastigotes, secreted proteins and/orpromastigotes within a Leishmania-infected sample. Preferably, thecontact time is sufficient to achieve a level of binding that is atleast 95% of that achieved at equilibrium between bound and unboundantigen. Those of ordinary skill in the art will recognize that the timenecessary to achieve equilibrium may be readily determined by assayingthe level of binding that occurs over a period of time. At roomtemperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20. Detectionreagent may then be added to the solid support. An appropriate detectionreagent is any compound that binds to the immobilized antibody-antigencomplex and that can be detected by any of a variety of means known tothose in the art. Preferably, the detection reagent contains a bindingagent (such as, for example, Protein A, Protein G, immunoglobulin,lectin or an antibody) conjugated to a reporter group. Preferredreporter groups include enzymes (such as horseradish peroxidase),substrates, cofactors, inhibitors, dyes, radionuclides, luminescentgroups, fluorescent groups and biotin. The conjugation of binding agentto reporter group may be achieved using standard methods known to thoseof ordinary skill in the art. Common binding agents may also bepurchased conjugated to a variety of reporter groups from many sources(e.g., Zymed Laboratories, San Francisco, Calif. and Pierce, Rockford,Ill.).

The detection reagent is then incubated with the immobilizedantibody-antigen complex for an amount of time sufficient to detect thebound antigen. An appropriate amount of time may generally be determinedfrom the manufacturer's instructions or by assaying the level of bindingthat occurs over a period of time. Unbound detection reagent is thenremoved and bound detection reagent is detected using the reportergroup. The method employed for detecting the reporter group depends uponthe nature of the reporter group. For radioactive groups, scintillationcounting or autoradiographic methods are generally appropriate.Spectroscopic methods may be used to detect dyes, luminescent groups andfluorescent groups. Biotin may be detected using avidin, coupled to adifferent reporter group (commonly a radioactive or fluorescent group oran enzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of Leishmania antigens in thesample, the signal detected from the reporter group that remains boundto the solid support is generally compared to a signal that correspondsto a predetermined cut-off value. This cut-off value is preferably theaverage mean signal obtained when the immobilized antibody is incubatedwith samples from an uninfected patient. In general, a sample generatinga signal that is three standard deviations above the mean is consideredpositive for Leishmania antigens and Leishmania infection. In analternate embodiment, the cut-off value is determined using a ReceiverOperator Curve, according to the method of Sackett et al., ClinicalEpidemiology: A Basic Science for Clinical Medicine, p. 106-7 (LittleBrown and Co., 1985). Briefly, in this embodiment, the cut-off value maybe determined from a plot of pairs of true positive rates (i.e.,sensitivity) and false positive rates (100%-specificity) that correspondto each possible cut-off value for the diagnostic test result. Thecut-off value on the plot that is the closest to the upper left-handcorner (i.e., the value that encloses the largest area) is the mostaccurate cut-off value, and a sample generating a signal that is higherthan the cut-off value determined by this method may be consideredpositive. Alternatively, the cut-off value may be shifted to the leftalong the plot, to minimize the false positive rate, or to the right, tominimize the false negative rate. In general, a sample generating asignal that is higher than the cut-off value determined by this methodis considered positive for Leishmania infection.

In one embodiment, the present invention provides an ELISA sandwichassay that may be effectively employed to detect the presence ofLeishmania amastigotes in samples taken from cutaneous lesions. In thisassay, an antibody specific for TSA (referred to as the captureantibody) is coated onto ELISA plates. After blocking, the plates areincubated with the biological sample, washed and then incubated with asecond anti-TSA antibody (referred to as the detection antibody), priorto being developed. In a preferred embodiment, the detection antibody isa TSA affinity purified rabbit polyclonal antibody that has been shownto detect TSA in low concentrations of solubilized amastigotes andpromastigotes, and the plate is developed using a goat anti rabbit IgGhorseradish peroxidase conjugate.

In a second embodiment, the assay is performed in a flow-through orlateral flow format, wherein the anti-TSA antibody is immobilized on amembrane such as nitrocellulose. In the flow-through test, antigenswithin the sample bind to the immobilized antibody as the sample passesthrough the membrane. A detection reagent then binds to theantibody-antigen complex as a solution containing the detection reagentflows through the membrane. The detection of bound detection reagent maythen be performed as described above. In the lateral flow format, oneend of the membrane to which antibody is bound is immersed in a solutioncontaining the biological sample. The sample migrates along the membranethrough a region containing the detection reagent, which preferablyincludes a calorimetric label, such as colloidal gold, and to the areaof immobilized capture antibody. Concentration of detection reagent atthe capture antibody indicates the presence of Leishmania amastigote inthe sample. Such tests can typically be performed with a very smallamount of biological sample.

FIG. 1 shows an exemplary dipstick which may be employed in theinventive methods to detect the presence of TSA in a biological sample.In this system, lines are striped (1 μl/cm) on a suitable membrane usingan automated biojet system (BioDot, Inc., Irvine, Calif.). The bottom,or test, line 10 consists of a TSA-specific capture antibody in asuitable buffer. One or more test lines can be employed depending on thenumber of antibodies to be incorporated. The top, or control, line 12 isused as an internal control to make sure that all the test componentsare working. In this embodiment, control line 12 is protein G or goatanti mouse or rabbit IgG. An anti-TSA antibody (“detection antibody”)conjugated to gold is employed as the detection reagent. Colloidal goldconsists of discrete, electron-dense, red-colored particles. Whenconcentrated on solid surfaces, these particles can be visuallyobserved. The detection reagent is dried onto a glass fiber pad, orconjugate pad, 14 and laminated below a membrane. Pads that can beeffectively employed as conjugate pad 14 include those available fromWhatman Inc. (Florham park, N.J.), such as the Whatman Rapid ReleasePad. A sample pad 16 is soaked in an appropriate buffer, dried andlaminated underneath pad 14. Pads that may be effectively employed assample pad 16 include those available from Ahlstrom Inc. An absorbenttop pad 18 is provided to remove excess fluid.

In use, the biological sample is applied onto the end of sample pad 16and followed with a chase buffer, preferably a phosphate based buffer.The sample mixes with the detection reagent and the resulting complexmoves laterally upward and binds to the capture antibody at test line 10if the sample contains TSA, causing test line 10 to turn red. Unboundcomplex will continue to travel upwards and will bind to the protein Gor goat anti-mouse or rabbit IgG at control line 12 depending on thenature of the antibody chosen for the mobile phase, causing control line12 to turn red. If a control line is not observed, the test isconsidered invalid.

Other formats for using monospecific antibodies to detect Leishmaniamajor infection in a sample will be apparent to those of ordinary skillin the art.

Assays employing the antibody WIC79.3 to detect the presence of L. majorsecreted proteins or promastigotes may be performed in a similar mannerto those employing anti-TSA antibodies.

As noted above, in certain embodiments, the inventive methods employantibodies, such as polyclonal or monoclonal antibodies, that arespecific for the Leishmania amastigote antigen TSA. The capture antibodyis a preferably a monoclonal antibody specific for TSA. In oneembodiment, the monoclonal antibody C11C is employed as the captureantibody. This antibody which, as detailed below, was developed againstLeishmania amastigotes and has been shown to react with TSA, isavailable from Dr. Matsumoto, University of Tokyo, Tokyo, Japan. Inalternative embodiments, the inventive methods employ the monoclonalantibody WIC79.3, which has been shown to be specific for L. majorlipophosphoglycan (LPG) (Kelleher et al. Proc. Natl. Acad. Sci. USA89:6-10, 1992; de Ibarra et al. Parasitology 85:523-31, 1982; Greenblattet al. J. Clin. Microbiol. 18:191-3, 1983; Handman et al. EMBO J. 1984;3(10): 2301-6, 1984), and is available from the Walter Reed ArmyInstitute of Research (Silver Spring, Md.).

Antibodies to purified, recombinant or synthesized antigens may beprepared by any of a variety of techniques known to those of ordinaryskill in the art. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In one such technique, animmunogen comprising the antigenic polypeptide is initially injectedinto any of a wide variety of mammals (e.g., mice, rats, rabbits, sheepand goats). In this step, the antigen, such as TSA, may serve as theimmunogen without modification. Alternatively, particularly forrelatively short polypeptides, a superior immune response may beelicited if the polypeptide is joined to a carrier protein, such asbovine serum albumin or keyhole limpet hemocyanin. The immunogen isinjected into the animal host, preferably according to a predeterminedschedule incorporating one or more booster immunizations, and theanimals are bled periodically. Polyclonal antibodies specific for theantigen may then be purified from such antisera by, for example,affinity chromatography using the antigen coupled to a suitable solidsupport.

Monoclonal antibodies specific for the antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and tested for bindingactivity against the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation and extraction.

The present invention also provides kits for use in the diagnosis ofcutaneous leishmaniasis. Such kits comprise: a dipstick as describedabove preferably pouched in a generally impermeable container, such asan aluminum foil container, with desiccant; a tube, or vessel,containing lysing buffer; a plastic rod/pestle for mixing skin lesionscrapings/biopsies with the lysing buffer to release amastigotes intosolution for testing; and a disposable transfer pipette for applyingsolution to the dipstick.

The following Examples are offered by way of illustration and not by wayof limitation.

Example 1 Preparation of Monoclonal Antibodies to Leishmania Amastigotes

Amastigotes for immunization were isolated from non-necrotic lesions atthe dorsal part of the tail base in L. major RM2-infected RAG^(2−/−)mice approximately 4 to 5 weeks after infection. The sonicatedamastigotes were centrifuged at 1,600 g for 30 min at 4° C. and theresulting supernatants were used as antigens. BALB/c mice were immunizedwith whole lysate of amastigotes with complete Freund's adjuvant. Afterthe first immunization, the mice were inoculated with the same dose ofamastigote homogenate every 3 weeks. The mice were given a final boostof amastigote homogenate 4 days before fusion. Hybrid cells wereproduced by fusing P3-X63-Ag8-U1 cells with spleen cells isolated fromthe immunized mice. Hybridomas were screened for production ofantibodies against amastigote antigens by ELISA. Limiting dilution wasperformed twice to obtain monoclonal hybridomas. One of the hybridomas,referred to as C11C, was inoculated into RAG^(2−/−) miceintraperitoneally. After 2 to 3 weeks ascites were harvested andcentrifuged, and the supernatant was collected. Immunoglobulins werepurified using Protein G.

Example 2 Preparation of Monoclonal and Polyclonal Antibodies toRecombinant TSA

Recombinant TSA expressed in E. coli was used as immunogen for preparingadditional monoclonal antibodies in mice and for raising a polyclonalantibody in rabbits. Supernatants from four monoclonal antibodies weretested for their reactivity against TSA and an unrelated T. cruziprotein as negative control. In addition, the rabbit polyclonal antibodywas affinity purified on a TSA sepharose column for use in ELISA andrapid test development.

Example 3 Reactivity of Antibodies Against L. Major Amastigotes andPromastigotes

Various antibodies, including C11C, were analyzed to determine theirreactivity with solubilized preparations of L. major promastigotes grownin culture, as well as with amastigotes derived from skin lesions ofmice infected with L. major. These studies enabled the selection ofantibodies/target antigens that reacted with both forms of L. major butwith particular emphasis on amastigotes, the form present in skinlesions. We initially analyzed antibodies to test their reactivity/titerwith a fixed concentration of antigen on an ELISA plate and then, usinga fixed dilution, determined what level of antigen could be detected inboth L. major promastigotes and amastigotes, the latter being derivedfrom skin lesions from infected mice. Polyclonal antibodies used inthese initial studies were against L. major amastigotes, TSA, LmSTI1(M15), HSP83 (JV91), HSP70 (JV8E), LACK antigen and a secretedpolyprotein from L. major (4A5). LmSTI1 and HSP83 are disclosed in USpublished patent application no. US 2002/0081320, the disclosure ofwhich is hereby incorporated by reference. Also evaluated were secretedproteins isolated from supernatants of cultured promastigotes of twostrains of L. major, L. tropica and L. mexicana provided by Walter ReedArmy Institute of Research (WRAIR).

Table 1 summarizes the reactivity of different antibodies withpromastigotes, amastigotes and secreted proteins from Leishmania spp.,with L. major LRC940 and L. major Pat2 being different isolates of L.major. As shown in Table 1, the C11C antibody and the rabbit anti-TSAantibody showed strong reactivity against both promastigotes andamastigotes as determined by ELISA. FIGS. 2-5 show the reactivity of thevarious antibodies to both promastigotes and to amastigotes derived frommouse skin lesions. The most reactive antibodies in both promastigotesand amastigotes were the rabbit anti-TSA antibody and the C11Cmonoclonal antibody to TSA, as well as the rabbit antibody to wholeamastigotes. Also strongly reactive were those to the heat shockproteins HSP70 and HSP83 (referred to as HSP 8E and HSP 91,respectively, in FIGS. 5A and B) which may be an alternative to TSA inassay development. LmST11, L. major polyprotein and LACK were onlyweakly reactive with both promastigotes and amastigotes.

TABLE 1 TSA Rabbit HSP70 HSP83 (C11C TSA anti (8E) (9I) LACK 4A5 Mab)(pAb) Amastigotes LmSTI1 (pAb) (pAb) (pAb) (pAb) Amastigotes ++++ ++++++++ + ++++ +++ + + Promastigotes ++++ ++++ +++ + ++++ ++++ + +Exoantigens L. major LRC940 ±* ±* − ± +++ ++ ± + L. major Pat2 ±* ±* − ±+++ ++ ± + L. mexicana FD ±* ±* − ± +++ ++ ± + L. tropica LRC590 ±* ±* −± +++ ++ ± +

Example 4 Sandwich ELISA for the Detection of TSA

Based on the above studies, a sandwich ELISA assay was established toassess the detectability of TSA in both promastigotes and amastigotes.The monoclonal antibody C11C was coated on the ELISA plate. Afterblocking, plates were incubated with either purified TSA or differentdilutions of promastigotes and amastigotes. After washing, plates wereincubated with TSA affinity purified rabbit anti-TSA antibody andfinally developed with a goat anti-rabbit IgG horse radish peroxidaseconjugate and TMB substrate. The data from these studies is shown inFIGS. 6 and 8. FIG. 6 shows a standard curve for detection of TSA usingthe sandwich ELISA. Ng levels of antigen were detectable in this assay.FIG. 8 shows the ability of this assay to detect TSA in culturedpromastigotes as well as amastigotes, but not in E. coli lysate and anunrelated T. cruzi recombinant protein. FIG. 7 shows the reactivity offour additional monoclonal antibodies raised to recombinant TSA thatcould potentially replace C11C or the polyclonal antibody to TSA. Noreactivity was detected when LmSTI1 was used as target antigen.

The TSA sandwich ELISA was performed as follows. Microtiter plates(Immulon 2, flat bottom, high binding) were coated at 200 ng/wellovernight at 4° C. in a carbonate/bicarbonate buffer pH 9.6. Afterwashing three times with phosphate buffered saline containing Tween 20(0.05%; PBST), the plates were blocked with PBS containing 1% bovineserum albumin and 0.05% Tween 20 for 1 hour. Antigens at desiredconcentration, or amastigotes or promastigotes solubilized in detergentcontaining buffer and diluted from known cell concentrations, were thenadded to the plate and incubated for 30 minutes at 37° C. After washingsix times in PBST, TSA affinity purified rabbit anti-TSA was added at1/1000 dilution and further incubated at 37° C. for 30 min. Plates wereagain washed six times with PBST and further incubated at 37° C. for 30minutes with a 1/10,000 dilution of goat anti rabbit IgG (H&L)-horseradish peroxidase labeled (Southern Biotech). After washing six times inPBST, the plate was developed with TMB substrate for 15 min at ambienttemperature and stopped with 1N H₂SO₄. Plates were immediately read atOD 450 nm.

Example 5 Lateral Flow Immunoassay for the Detection of TSA

A lateral flow immunoassay was constructed as shown in FIG. 1. C11Cantibody was coated on the membrane at a concentration of 1 mg/ml as thetest line. Colloidal gold conjugate was prepared by diluting rabbitanti-TSA and adding to gold salt. After incubation, 5% BSA was added asa blocking reagent. Following mixing, the gold-antibody conjugate wasconcentrated by spinning down and discarding the supernatant. The goldwas diluted to the appropriate OD at 520-540 nm using gold suspensionbuffer and stabilizers, and used at an OD of 12. The antibody-goldconjugate was then sprayed onto the conjugate pad. The control line wasrecombinant Protein A sprayed at a concentration of 1 mg/ml. Antigens orsolubilized amastigotes or promastigotes (20 ul) were applied to thesample pad followed by 3 drops of chase buffer (PBS plus 0.1% sodiumazide). The lateral flow was evaluated with varying dilutions of TSA, L.major amastigotes and promastigotes, and secreted proteins produced inculture from various leishmania spp. FIG. 9 shows the reactivity ofamastigotes and promastigotes in a lateral flow assay for TSA asdescribed in Example 5.

Example 6 Sandwich ELISA and Lateral Flow Immunoassay for IdentifyingWIC79.3 Antigen in L. major Promastigotes and Secreted Proteins

A monoclonal antibody known as WIC79.3, (available from WRAIR) wereevaluated for use in assays. This antibody has been shown to be specificfor L. major lipophosphoglycan (LPG) (Kelleher et al. Proc. Natl. Acad.Sci. USA 89:6-10, 1992; de Ibarra et al. Parasitology 85:523-31, 1982;Greenblatt et al. J. Clin. Microbiol. 18:191-3, 1983; Handman et al.EMBO J. 1984; 3(10): 2301-6, 1984).

Initial studies using the WIC79.3 monoclonal antibody with secretedproteins from various Leishmania spp. as solid phase in an ELISA assayindicated that this antibody detects an antigen in L. major secretedproteins, as well as in whole L. major promastigotes, more effectivelythan in L. tropica and L. mexicana. In addition, this antigen was notdetectable in L. major amastigotes. Further analysis was then performedusing a WIC79.3 sandwich ELISA in which WIC79.3 was used as both thesolid phase antigen and as detection antibody to detect secretedproteins. In this study the antibody was biotinylated and the assaydeveloped using avidin HRP and TMB as substrate. The data is shown inFIG. 10.

A lateral flow assay using the WIC79.3 antibody was also developed usingthe techniques described above. As shown in FIG. 11, this assay wasfound to be capable of detecting L. major exoantigens and thus has thepotential to be used for specific identification of secreted antigens insamples taken from skin lesions.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

SEQ ID NO: 1 and 2 are set out in the attached Sequence Listing. Thecodes for polynucleotide and polypeptide sequences used in the attachedSequence Listing confirm to WIPO Standard ST.25 (1988), Appendix 2.

All references disclosed herein, including patent references andnon-patent references, are hereby incorporated by reference in theirentirety as if each was incorporated individually.

1. A method for detecting cutaneous leishmaniasis in a biologicalsample, comprising: (a) contacting the biological sample with anantibody specific for a polypeptide of SEQ ID NO: 1; and (b) detectingin the biological sample the presence of antigens that bind to theantibody, thereby detecting cutaneous leishmaniasis in the biologicalsample. 2-4. (canceled)
 5. The method of claim 1, wherein the antibodyis a monoclonal antibody.
 6. (canceled)
 7. The method of claim 1,wherein the presence of antigens that bind to the antibody is detectedby means of ELISA.
 8. A method for detecting cutaneous leishmaniasis ina biological sample, comprising: (a) contacting the biological samplewith a first antibody specific for a polypeptide of SEQ ID NO: 1 to forman antibody-polypeptide conjugate, wherein the first antibody is labeledwith a reporter agent; (b) contacting the antibody-polypeptide conjugatewith a second antibody specific for a polypeptide of SEQ ID NO: 1 inorder to capture the antibody-polypeptide conjugate; and (c) detectingthe presence of the captured antibody-polypeptide conjugate, therebydetecting cutaneous leishmaniasis in the biological sample. 9-13.(canceled)
 14. The method of claim 8, wherein the first antibody is apolyclonal antibody.
 15. The method of claim 8, wherein the secondantibody is a monoclonal antibody.
 16. The method of claim 15, whereinthe second antibody is C11C.
 17. A dipstick for use in the method ofclaim
 8. 18. A dipstick for the detection of cutaneous leishmaniasis,comprising: (a) a lateral flow membrane; (b) a first area positioned ata lower end of the lateral flow membrane for receiving a test sample,wherein the first area comprises a first antibody specific for apolypeptide of SEQ ID NO: 1, the antibody being labeled with a reporteragent; (c) a second area positioned at an upper end of the lateral flowmembrane comprising an immobilized control polypeptide; and (d) a thirdarea positioned between the first and second areas, wherein the thirdarea comprises an immobilized second antibody specific for a polypeptideof SEQ ID NO:
 1. 19. The dipstick of claim 18, wherein the reporteragent is a colorimetric or fluorescent indicator.
 20. The dipstick ofclaim 18, wherein the reporter agent is colloidal gold or a fluorescentdye.
 21. The dipstick of claim 18, wherein the first and secondantibodies are specific for a conformational epitope of the polypeptideof SEQ ID NO: 1 found in L. major promastigotes. 22-24. (canceled)
 25. Adiagnostic kit comprising a dipstick of claim
 17. 26. The diagnostic kitof claim 25, further comprising (a) a vessel containing lysing buffer;(b) a rod for mixing a biological sample with the lysing buffer toprovide a test solution; and (c) a pipette for applying the testsolution to the dipstick.
 27. (canceled)
 28. A method for detectingcutaneous leishmaniasis in a biological sample, comprising: (a)contacting the biological sample with the antibody WIC79.3; and (b)detecting in the biological sample the presence of antigens that bind tothe antibody, thereby detecting cutaneous leishmaniasis in thebiological sample. 29-32. (canceled)
 33. A method for detectingcutaneous leishmaniasis in a biological sample, comprising: (a)contacting the biological sample with the antibody WIC79.3 to form anantibody-polypeptide conjugate, wherein the WIC79.3 antibody is labeledwith a reporter agent; (b) contacting the antibody-polypeptide conjugatewith WIC79.3 in order to capture the antibody-polypeptide conjugate; and(c) detecting the presence of the captured antibody-polypeptideconjugate, thereby detecting cutaneous leishmaniasis in the biologicalsample. 34-38. (canceled)
 39. A dipstick for use in the method of claim33.
 40. A dipstick for the detection of cutaneous leishmaniasis,comprising: (a) a lateral flow membrane; (b) a first area positioned ata lower end of the lateral flow membrane for receiving a test sample;wherein the first area comprises the antibody WIC79.3 labeled with areporter agent; (c) a second area positioned at an upper end of thelateral flow membrane comprising an immobilized control polypeptide; and(d) a third area positioned between the first and second areas, whereinthe third area comprises non-labeled antibody WIC 79.3. 41-42.(canceled)
 43. A diagnostic kit comprising a dipstick of claim
 39. 44.The diagnostic kit of claim 43, further comprising: (a) a vesselcontaining lysing buffer; (b) a rod for mixing a biological sample withthe lysing buffer to provide a test solution; and (c) a pipette forapplying the test solution to the dipstick.
 45. (canceled)